Minimal residual disease detected by droplet digital PCR in peripheral blood stem cell grafts has a prognostic impact on high-risk neuroblastoma patients

Abstracts More than half of high-risk neuroblastoma (NB) patients have experienced relapse due to the activation of chemoresistant minimal residual disease (MRD) even though they are treated by high-dose chemotherapy with autologous peripheral blood stem cell (PBSC) transplantation. Although MRD in high-risk NB patients can be evaluated by quantitative PCR with several sets of neuroblastoma-associated mRNAs (NB-mRNAs), the prognostic significance of MRD in PBSC grafts (PBSC-MRD) is unclear. In the present study, we collected 20 PBSC grafts from 20 high-risk NB patients and evaluated PBSC-MRD detected by droplet digital PCR (ddPCR) with 7NB-mRNAs (CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH mRNA). PBSC-MRD in 11 relapsed patients was significantly higher than that in 9 non-relapsed patients. Patients with a higher PBSC-MRD had a lower 3-year event-free survival (P = 0.0148). The present study suggests that PBSC-MRD detected by ddPCR with 7NB-mRNAs has a prognostic impact on high-risk NB patients.


Introduction
Neuroblastoma (NB) is one of the most common solid tumors in children. It originates from neural crest-derived sympathoadrenal progenitors [1,2,3] and shows extreme heterogeneity, ranging from spontaneous regression to malignant progression [4,5]. Approximately half of newly diagnosed NB patients are assigned to a high-risk group and total NB patients account for~15% of pediatric cancer-associated deaths. High-risk NB patients are treated by the standard regimen consisting of induction chemotherapy, high-dose chemotherapy with autologous peripheral blood stem cell (PBSC) transplantation, surgery, radiotherapy, and immunotherapy [6,7]. Although as many as 20% of high-risk NB patients have residual disease that is refractory or progressive [7,8], the rest of the patients usually achieve remission during induction chemotherapy but potentially have the minimal residual disease (MRD) that causes relapse after completion of the standard regimen [9]. Consequently, more than half of high-risk NB patients still have experienced progression or relapse with a long-term survival rate of around 50% [10].
Patients with relapsed NB have a dismal outcome with less than a 10% rate of long-term survival [11].
To identify MRD in NB patients, a set of neuroblastoma-associated mRNAs (NB-mRNAs) are commonly quantitated in bone marrow (BM), peripheral blood (PB), or PBSC samples due to the lack of recurrent genomic aberrations in NB cells [12,13]. Several MRD assays quantitating different NB-mRNAs by quantitative PCR (qPCR) or droplet digital PCR (ddPCR) have been shown to possess a significant prognostic value of MRD in BM samples (BM-MRD) for high-risk NB patients [14,15,16,17,18]. Although we and others have shown the significantly correlated MRD levels between BM and PB samples [17,18,19], the prognostic significance of MRD in PB and PBSC samples (PB-MRD and PBSC-MRD) is still unclear.
Because PB has no or fewer detectable tumor cells by immunocytology even when BM is positive in high-risk NB patients, there may be a reduced risk of contamination with tumor cells in PBSC grafts than in BM grafts [20]. PBSC grafts become the first choice for autologous stem cells to restore hemopoiesis after high-dose chemotherapy in the current protocol for high-risk NB patients [7]. However, it remains elusive whether reinfusion of PBSC grafts contaminated with tumor cells can cause relapse and impact the outcome of high-risk NB patients [21]. There are conflicting reports on a prognostic impact of qPCR-based PBSC-MRD monitoring. PBSC-MRD detected by qPCR with B4GALNT1 or TH mRNA was not statistically associated with unfavorable outcomes [22,23,24]. In contrast, high PBSC-MRD detected by qPCR with CHGA, DCX, DDC, PHOX2B, or TH mRNA predicted poor outcome [21,25]. Furthermore, the opposite prognostic impact of qPCR-based PBSC-MRD monitoring was reported for the same cohort. Although PBSC-MRD detected by qPCR with the commonly used adrenergic (ADRN) NB-mRNAs (CHRNA3, DBH, DDC, PHOX2B, or TH mRNA) was not correlated with the outcome, the newly identified mesenchymal (MES) NB-mRNAs (POSTN or PRRX1 mRNA) enabled PBSC-MRD to be significantly associated with poor event-free survival (EFS) and overall survival (OS) [26,27].
Although we have shown that ddPCR-based MRD monitoring with 7NB-mRNAs (CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH mRNAs) has a significant prognostic value for BM samples [18], it remains to be determined for PBSC grafts. In the present study, we performed ddPCR-based MRD monitoring in 20 PBSC grafts and evaluated its clinical impact on high-risk NB patients.

NB patients and samples
Twenty high-risk NB patients according to the Children's Oncology Group (COG) Neuroblastoma Risk Stratification System [5,28] or the International Neuroblastoma Risk Group (INRG) Classification System [29] were included in this study.

7NB-mRNAs ddPCR assay
7NB-mRNAs ddPCR assay was carried out as described previously [18,19,31]. Briefly, total RNA was extracted from PBSC samples using a TRIzol Plus RNA purification kit (Life Technologies, Carlsbad, CA). cDNA was synthesized from 1 μg or 0.5 μg total RNA with a Quantitect reverse transcription kit (Qiagen, Valencia, CA, USA) and stored at À80 C until use. The expression of 7 NB-mRNAs (CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH) and a reference gene mRNA (HPRT1) was quantitated by a QX200 ddPCR system (Bio-Rad Laboratories, Hercules, CA) according to the digital MIQE (Minimum Information for Publication of Quantitative Digital PCR Experiments) guideline [32,33]. The level of 7NB-mRNAs (combined signature) was defined as the weighted sum of 7 relative copy numbers (level of each NB-mRNA), in which the reciprocal of 90 percentile in non-NB control PB samples [18] was used for the weighting for each NB-mRNA.

Statistical analysis
The differences in the levels of 7NB-mRNAs between two sample populations were evaluated by the Mann-Whitney U-test. The prognostic value of the levels of 7NB-mRNAs was assessed using the receiver operator characteristic (ROC) analysis. The area under curve (AUC) was interpreted 0.50-0.69 as "low accuracy", 0.70-0.89 as "moderate accuracy", and "0.90-1.00" as high accuracy [34]. EFS was defined as the time from the date of diagnosis to the date of relapse or progression. OS was defined as the time from the date of diagnosis to the date of death. EFS and OS were analyzed by the Kaplan-Meier method and log-rank test, and the observation period was censored at 120 months. Reported P values were two-sided and P < 0.05 was considered statistically significant. EZR (version 1.35, www.jichi.ac.jp/saitama-sct/SaitamaHP. files/statmedEN.html; Saitama Medical Center, Jichi Medical University, Saitama, Japan), a modified version of R commander [35], was used for statistical analyses.

Patient and sample characteristics
Twenty patients included in the present study were all high-risk patients. As summarized in Table 1

Association between the level of 7NB-mRNAs in PBSC grafts and relapse of high-risk NB patients
Although the level of 7NB-mRNAs in PBSC grafts was not so high as in BM and PB samples at diagnosis [18], it was significantly higher in PBSC grafts harvested from 11 relapsed patients than in those from 9 non-relapsed patients (Figure 3a). The ROC curve was then plotted for the level of 7NB-mRNAs (Figure 3b). It estimated an AUC of 0.768 with significant accuracy (>0.7), showing that a higher level of 7NB-mRNAs in PBSC grafts predicted relapse of high-risk NB patients.

Discussion
In the present study, we performed ddPCR-based PBSC-MRD monitoring in 20 PBSC grafts with 7NB-mRNAs and revealed that higher PBSC-MRD was significantly associated with poorer EFS in high-risk NB patients. To our knowledge, this is the first study to demonstrate a significant prognostic value of ddPCR-based PBSC-MRD for high-risk NB patients.
A clinical significance of PBSC-MRD remained to be established for high-risk NB patients over the last decades. PBSC-MRD detected by qPCR with B4GALNT1, CHRNA3, DBH, DDC, PHOX2B, or TH mRNA was shown to be not statistically associated with outcome [22,23,24,26], while higher PBSC-MRD detected by qPCR with CHGA, DCX, DDC, PHOX2B, or TH mRNA was reported to be correlated with worse outcome [21,25]. Compared to qPCR, ddPCR is more sensitive to detect specific nucleic acids and potentially provides more accurate and reproducible quantification of low-level of NB-mRNAs. In the present study, we revealed that higher PBSC-MRD detected by ddPCR with CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH mRNAs was associated with lower EFS (Figure 4) [18]. Although it is tempting to speculate the superiority of ddPCR for PBSC-MRD monitoring in high-risk NB patients, further validation of ddPCR-based PBSC-MRD monitoring using a larger cohort will be required.
Although NB cells present with very diverse phenotypes, recent studies explored the transcriptomic and epigenetic landscapes of NB cells together with neural crest (NC) cells and identified two super-enhancers (SEs) consisting of dense clusters of enhancers that underlay cell and lineage identity [36,37,38]. Based on these SEs, NB cells were classified into two distinct cell states: a neuronal sympathetic ADRN state associated with ADRN NB-mRNAs expression and an undifferentiated and NC-like MES state associated with MES NB-mRNAs expression. These ADRN and MES cells corresponded to the previously described neuroblast (N)-type and substrate-adherent (S)-type cells and had the ability to transdifferentiate or interconvert into each other. In comparison with ADRN cells, MES cells were reported to be more resistant to standard chemotherapeutic drugs such as cisplatin, doxorubicin, and etoposide used to treat NB patients and enriched in post-treatment and recurrent tumors. As MRD was defined as residual tumor cells following local and  systemic treatment [39], MES NB-mRNAs were identified and examined for PBSC-MRD detection of high-risk NB patients [27]. Using the same cohort, PBSC-MRD detected by qPCR with MES NB-mRNAs (POSTN or PRRX1 mRNA), but not ADRN NB-mRNAs (CHRNA3, DBH, DDC, PHOX2B, or TH mRNA), was reported to be significantly associated with EFS and OS of high-risk NB patients [26,27]. In the present study, PBSC-MRD was detected by ddPCR with different ADRN NB-mRNAs (CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH mRNAs) and was significantly associated with EFS of high-risk NB patients. Further studies using a larger cohort will be required to determine the optimal set of NB-mRNAs (ADRN NB-mRNAs, MES NB-mRNAs, or their combination) for PBSC-MRD detection in high-risk NB patients.
In conclusion, we have revealed that PBSC-MRD detected by ddPCR with 7NB-mRNAs has a significant prognostic value for high-risk NB patients.

Declarations
Author contribution statement Noriyuki Nishimura: Conceived and designed the experiments, Analyzed and interpreted the data, and Wrote the paper.

Data availability statement
Data included in article/supp. material/referenced in article.

Declaration of interest's statement
The authors declare the following conflict of interests: Noriyuki Nishimura received institutional research funding from Sysmex Corporation to Kobe University.

Additional information
No additional information is available for this paper.